Continued support is requested to analyze the components and regulators of a signaling pathway that suppresses integrin activation, a process central to blood cell migration and platelet aggregation. This pathway is initiated activation, a process central to blood cell migration and platelet aggregation. This pathway is initiated by activated H-Ras GTPase or its effector, Raf-1 kinase, PEA-15, a death effector domain-containing protein, regulates this pathway by reversing the suppressive effect of H- Ras. PEA-15 also binds to ERK + MAP kinase and phospholipase D (PLD), and causes cytoplasmic sequestration of ERK. The applicant hypothesizes that one or both of these biochemical functions of PEA-15 are required for its effects on integrins. To test this hypothesis, interactions of PEA-15 with ERK and PLD will be mapped. This information will be used to produce point mutations that selectively disrupt PEA-15 interactions with ERK or PLD and to assess the role of these interactions in the reversal of suppression of integrin activation. The mechanism by which PEA-15 regulates ERK subcellular localization will also be assessed in order to test the role of ERK localization in the integrin suppressor pathway. Furthermore, the small GTPase, R-Ras, reverses H-Ras suppression of integrin activation via an apparently novel effector. By making chimeras between structurally related GTPases (R-Ras and H-Ras) with opposing effects on integrin activation, the applicant will analyze the mechanisms of suppression and reversal of suppression. The chimeras will be used to whether differential R-Ras and H-Ras subcellular localization accounts for their opposing effects on integrins. In addition, these chimeras and previously identified effector loop mutants will be used to search for novel effectors of Ras GTPases that regulate integrin. Collaboration with Projects and 3 will explore the subcellular localization of novel effectors in megakaryocytes, T lymphocytes and endothelial cells and determine their roles in integrin functions. These studies will provide fundamental insights into pathways that control the activation of integrins in cells of the blood and vasculature and may serve to identify novel therapeutic targets for diseases of hemostasis and thrombosis.